Cell survival depends on the ability to respond to stress signals from the extracellular environment. Diverse stress signals induce the expression of specific genes that function in the physiologic response to the stress. In the absence of stress, expression of many of these genes is maintained at a minimal level. We have found in the yeast S. cerevisiae, a model eukaryotic system, that the basal expression of many stress-induced genes is minimized by a novel mechanism - premature transcriptional termination, or transcriptional attenuation. Genes induced by cell wall stress require the MAP kinase Mpk1 to carry out two separate steps in the transcription process, neither of which requires its protein kinase activity. The first is to recruit a transcription factor to promoters of target genes. The second involves blocking attenuation, which occurs within the promoter-proximal region of target genes under non-inducing conditions. Attenuation is mediated by the Sen1 termination complex and is blocked by the translocation of Mpk1 to the elongating RNA polymerase (Pol II). Under inducing conditions, gene expression depends upon the relief of attenuation. For Mpk1-induced genes, this happens through the association of Mpk1 with the elongation factor Paf1, which blocks the recruitment of the Sen1 complex to Pol II. This interaction is conserved in the human ortholog of Mpk1, ERK5, suggesting that regulated transcriptional attenuation operates in humans. Based on our preliminary findings, we propose that a wide variety of stress-induced genes are silenced by transcriptional attenuation under non-inducing conditions and that a constellation of transcription factors are likely to relieve attenuation under inducing conditions through interactions with the Paf1C (a complex containing Paf1). The long-term objective of this project is to provide a novel approach to blocking the expression of specific genes, or groups of genes, by inhibiting relief of transcriptional attenuation. We propose to elucidate the mechanisms that regulate transcriptional attenuation and the degree to which various stresses use similar or different attenuation-relief factors to regulate a variety of target genes. One immediate goal will be to determine if other MAP kinases that respond to different signals also function as attenuation-relief factors. Another project will identify non-MAP kinase attenuation- relief factors that allow the induction of a variety of stress-induced genes we have found to be under attenuation control. A third goal will be to understand the role of the Paf1C in the recruitment of the Sen1 termination complex to Pol II. Overall, these studies will yield a mechanistic understanding of regulated transcriptional attenuation and reveal the ubiquity of the process in yeast, which will inform subsequent studies on human cells. PUBLIC HEALTH RELEVANCE: We have discovered a novel mechanism for the control of eukaryotic gene expression - premature transcriptional termination, or transcriptional attenuation. The long-range goal of this project is to provide a novel approach to blocking the expression of specific genes, or groups of genes, by stimulating transcriptional attenuation. We envision the development of small molecule drugs that result in gene silencing by constitutive attenuation, which may be applied to novel anti- fungal therapies, and may extend to therapeutic human gene silencing.